The two structurally related cytokines tumour necrosis factor (TNF-.alpha.) and lymphotoxin (TNF-.beta.) were originally discovered as a result of their cytotoxic in vitro activity against tumour cells and their ability to induce haemorrhagic necroses of tumours in a mouse model. The cloning of their cDNAs and their expression in E. coli have made these proteins available in virtually unlimited quantities and have made it possible to develop highly specific antibodies and sensitive immunoassays. There is a wealth of information on the biological activities of these proteins, their physiological roles as pleotropic mediators of inflammatory processes and their participation in pathological conditions. In particular, an increased production of TNF-.alpha. has been linked with the pathogenesis of, for example, septic shock, tissue damage in the "Graft-versus-host" disease and cerebral malaria and cachexia (Beutler, 1988; Paul and Ruddle, 1988; Beutler and Cerami, 1989).
The possible undesirable effects of TNF-.alpha. have led to a search for natural inhibitors of this cytokine. A protein which binds to TNF-.alpha. and thereby inhibits the activity thereof was originally identified in the urine of patients with kidney failure (Peetre et al., 1988) and fever patients (Seckinger et al., 1988). This protein with an apparent molecular weight of about 30 kDa was purified in order to homogenise it, partially sequenced (EP-A2 308 378) and the cDNA was cloned (Olsson et al., 1989; Engelmann et al., 1989; Himmler et al., 1990; Schall et al., 1990). The structure of the cDNA showed that this protein, designated TNF-BP, is the extracellular fragment of a TNF receptor (TNF-R). (It was assumed that this fragment is released by proteolytic cleaving.) These results were confirmed by isolation of the intact membrane receptor for TNF-.alpha., which was carried out independently by other working groups (Loetscher et al., 1990). The entire receptor protein consists of 455 amino acids (55-60 kDa); TNF-BP makes up the majority of the extracellular domain of the receptor and contains all three N-glycosylation sites. It was found that TNF-BP isolated from urine is heterogeneous at the N-terminus as a result of proteolytic cleaving and is therefore a mixture of two molecular forms consisting of 161 amino acids (main fraction) or 172 amino acids (Himmler et al., 1990). Recently, the existence of a second TNF-binding protein was demonstrated, which is a fragment of a second TNF receptor type having a higher molecular weight (75-80 kDa; Engelmann et al., 1990a; Smith et al., 1990; Kohno et al., 1990). The two receptors and binding proteins were then designated TNF-R I/TNF-BP I and TNF-R II/TNF-BP II (for the 60 kDa and 80 kDa receptors, respectively). Sequence comparison showed that the two proteins are structurally related; in particular, the number and distribution of the cysteine groups is very similar. However, the two receptors differ from each other immunologically (Engelmann et al., 1990a; Brockhaus et al., 1990).
The human 60 kDa TNF-receptor plays an essential part in TNF.alpha.-signal transmission. The activity of the receptor is subjected to several regulatory effects on a protein basis: the treatment of cells with phorbol esters or other activators of protein kinase C results in a rapid decrease in the number of cellular binding sites for TNF-.alpha.. This is linked with the release of the extracellular part of the receptor, corresponding to TNF-BP I, by proteolytic cleaving. Similar effects, albeit with different kinetics, are caused by various other substances, particularly by the physiological ligands TNF-.alpha. and TNF-.beta.. The cleavage sites for the protease on human and rat TNF-R I are conserved; they differ in structure from the specificity of all known proteases. It is therefore probable that a highly specific proteolytic enzyme is part of a regulating circuit which controls the sensitivity of cells to TNFs; the exact mechanism of these events is not yet known. Recently this phenomenon has also been observed in vivo: it was found that the administration of TNF-.alpha. to cancer patients resulted in a significant increase of TNF-BP I in the serum (Lantz et al., 1990a).
The concentration of TNF-BP I in culture residues or body fluids is therefore an indicator of the activation of the TNF receptor system in vitro or in vivo as a result of interactions with the ligands or transmodulation by other mediators; the TNF-BP I concentration in body fluids can thus be regarded as a useful marker for various diseases.
There was therefore a need for efficient and sensitive methods of detecting TNF-BP I and for kits which can be used for such detection methods.
Monoclonal antibodies against TNF-R I have been described which were prepared by immunising with the solubilised receptor (Brockhaus et al., 1990; EP A2 334 165; Thoma et al., 1990) or with purified TNF-BP I (Engelmann et al., 1990b); however, it was not shown that the antibodies are suitable for use in immunoassays for TNF-BP I.
Lantz et al. (1990a) have developed a competitive ELISA in which, in a three-step test method, test plates coated with TNF-BP I, polyclonal rabbit antibodies against TNF-BP I, biotin-labelled goat antibodies against rabbit immunoglobulin and avidin-coupled alkaline phosphatase were used. By means of this assay, the presence of TNF-BP I in serum from normal donors was detected and increased concentrations of TNF-BP I were detected in sera from patients suffering from kidney failure or cancer patients who had been treated with TNF-.alpha..
EP A1 412 486 describes monoclonal antibodies against TNF-BP I. One of these antibodies was used in a sandwich ELISA as a coating antibody: polyclonal rabbit anti-TNF-BP I antibody was used as the second antibody and polyclonal goat anti-rabbit antibody was used as the third, enzyme-coupled antibody.
The known assays are complicated in their structure and procedure required and moreover the use of polyclonal antibodies involves the use of animals, which is something which is increasingly to be avoided.